Whole Lung Tissue vs. BAL Fluid 16S Sequencing Analysis
Jenn Baker
June 24, 2020
FALSE R version 4.0.2 (2020-06-22)
FALSE Platform: x86_64-apple-darwin17.0 (64-bit)
FALSE Running under: macOS Catalina 10.15.6
FALSE
FALSE Matrix products: default
FALSE BLAS: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRblas.0.dylib
FALSE LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib
FALSE
FALSE locale:
FALSE [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
FALSE
FALSE attached base packages:
FALSE [1] stats graphics grDevices utils datasets methods base
FALSE
FALSE other attached packages:
FALSE [1] cbmbtools_0.0.0.9034 vegan_2.5-6 lattice_0.20-41
FALSE [4] permute_0.9-5 ggpubr_0.4.0 RColorBrewer_1.1-2
FALSE [7] knitr_1.29 scales_1.1.1 tidyselect_1.1.0
FALSE [10] forcats_0.5.0 stringr_1.4.0 dplyr_1.0.1
FALSE [13] purrr_0.3.4 readr_1.3.1 tidyr_1.1.1
FALSE [16] tibble_3.0.3 ggplot2_3.3.2 tidyverse_1.3.0
FALSE
FALSE loaded via a namespace (and not attached):
FALSE [1] httr_1.4.2 jsonlite_1.7.0 splines_4.0.2 carData_3.0-4
FALSE [5] modelr_0.1.8 assertthat_0.2.1 blob_1.2.1 cellranger_1.1.0
FALSE [9] yaml_2.2.1 pillar_1.4.6 backports_1.1.8 glue_1.4.1
FALSE [13] digest_0.6.25 ggsignif_0.6.0 rvest_0.3.6 colorspace_1.4-1
FALSE [17] htmltools_0.5.0 Matrix_1.2-18 pkgconfig_2.0.3 broom_0.7.0
FALSE [21] haven_2.3.1 openxlsx_4.1.5 rio_0.5.16 mgcv_1.8-31
FALSE [25] generics_0.0.2 car_3.0-9 ellipsis_0.3.1 withr_2.2.0
FALSE [29] cli_2.0.2 magrittr_1.5 crayon_1.3.4 readxl_1.3.1
FALSE [33] evaluate_0.14 fs_1.5.0 fansi_0.4.1 nlme_3.1-148
FALSE [37] MASS_7.3-51.6 rstatix_0.6.0 xml2_1.3.2 foreign_0.8-80
FALSE [41] tools_4.0.2 data.table_1.13.0 hms_0.5.3 lifecycle_0.2.0
FALSE [45] munsell_0.5.0 reprex_0.3.0 cluster_2.1.0 zip_2.0.4
FALSE [49] compiler_4.0.2 rlang_0.4.7 grid_4.0.2 rstudioapi_0.11
FALSE [53] rmarkdown_2.3 codetools_0.2-16 gtable_0.3.0 abind_1.4-5
FALSE [57] DBI_1.1.0 curl_4.3 R6_2.4.1 lubridate_1.7.9
FALSE [61] stringi_1.4.6 parallel_4.0.2 Rcpp_1.0.5 vctrs_0.3.2
FALSE [65] dbplyr_1.4.4 xfun_0.16
FALSE
FALSE Kruskal-Wallis rank sum test
FALSE
FALSE data: Gene_16S_copies_per_mL by Sample_Type
FALSE Kruskal-Wallis chi-squared = 29.276, df = 6, p-value = 5.394e-05
FALSE
FALSE
FALSE Pairwise comparisons using Wilcoxon rank sum exact test
FALSE
FALSE data: ddpcr$Gene_16S_copies_per_mL and ddpcr$Sample_Type
FALSE
FALSE NTC Iso_Ctrl PBS Syringe_Rinse Homog_Ctrl BAL
FALSE Iso_Ctrl 0.64000 - - - - -
FALSE PBS 0.58947 0.40000 - - - -
FALSE Syringe_Rinse 0.42353 0.03333 0.40000 - - -
FALSE Homog_Ctrl 0.58947 0.16667 0.42353 0.40000 - -
FALSE BAL 0.22238 0.00839 0.31818 0.27082 0.90909 -
FALSE Lung 0.00839 0.00262 0.07955 0.00839 0.07955 0.00023
FALSE
FALSE P value adjustment method: BHQuality Checks
III. Confirmation of Sufficient Reads
3.1 MiSeq Reads by Sample Type
## 3.2 OTU Counts by Sample Type
IV. Principal Component Analysis
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.df.wbn ~ otu.df.lungsamp.sampctrls$RA_Groups, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2
FALSE otu.df.lungsamp.sampctrls$RA_Groups 2 3.8879 1.94394 2.3806 0.16554
FALSE Residuals 24 19.5980 0.81658 0.83446
FALSE Total 26 23.4859 1.00000
FALSE Pr(>F)
FALSE otu.df.lungsamp.sampctrls$RA_Groups 9.999e-05 ***
FALSE Residuals
FALSE Total
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.bl ~ adonis.otudf.bl$Sample_Type, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.bl$Sample_Type 1 2.4484 2.4484 3.1334 0.15563 9.999e-05 ***
FALSE Residuals 17 13.2838 0.7814 0.84437
FALSE Total 18 15.7323 1.00000
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.wn ~ adonis.otudf.wn$RA_Groups, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.wn$RA_Groups 1 2.4698 2.46976 3.3064 0.18061 2e-04 ***
FALSE Residuals 15 11.2046 0.74697 0.81939
FALSE Total 16 13.6744 1.00000
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.bn ~ adonis.otudf.bn$Organ, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.bn$Organ 1 0.9148 0.91483 0.99522 0.05856 0.4649
FALSE Residuals 16 14.7076 0.91922 0.94144
FALSE Total 17 15.6224 1.00000
FALSE
FALSE Call:
FALSE adonis(formula = otu.good.lungsamp.isoctrls_hel ~ otu.df.lungsamp.isoctrls$Sample_Type, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2
FALSE otu.df.lungsamp.isoctrls$Sample_Type 3 5.0693 1.6898 2.0376 0.1846
FALSE Residuals 27 22.3910 0.8293 0.8154
FALSE Total 30 27.4603 1.0000
FALSE Pr(>F)
FALSE otu.df.lungsamp.isoctrls$Sample_Type 9.999e-05 ***
FALSE Residuals
FALSE Total
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.il ~ adonis.otudf.il$RA_Groups, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.il$RA_Groups 1 2.6804 2.68040 3.4196 0.15253 9.999e-05 ***
FALSE Residuals 19 14.8927 0.78383 0.84747
FALSE Total 20 17.5731 1.00000
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.ib ~ adonis.otudf.ib$RA_Groups, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.ib$RA_Groups 1 1.2087 1.20866 1.3141 0.06165 0.06999 .
FALSE Residuals 20 18.3957 0.91978 0.93835
FALSE Total 21 19.6044 1.00000
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = otu.good.lungsamp.seqctrls_hel ~ otu.df.lungsamp.seqctrls$Sample_Type, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2
FALSE otu.df.lungsamp.seqctrls$Sample_Type 3 7.919 2.63959 3.2847 0.16193
FALSE Residuals 51 40.984 0.80361 0.83807
FALSE Total 54 48.903 1.00000
FALSE Pr(>F)
FALSE otu.df.lungsamp.seqctrls$Sample_Type 9.999e-05 ***
FALSE Residuals
FALSE Total
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.sl ~ adonis.otudf.sl$RA_Groups, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.sl$RA_Groups 1 4.146 4.1459 5.1613 0.10717 9.999e-05 ***
FALSE Residuals 43 34.541 0.8033 0.89283
FALSE Total 44 38.687 1.00000
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.sb ~ adonis.otudf.sb$RA_Groups, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.sb$RA_Groups 1 2.069 2.06931 2.3933 0.05159 0.0006999 ***
FALSE Residuals 44 38.044 0.86463 0.94841
FALSE Total 45 40.113 1.00000
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = otu.good.tong.lungsamp_hel ~ otu.df.tong.lungsamp$Sample_Type, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE otu.df.tong.lungsamp$Sample_Type 2 3.9663 1.98317 2.6355 0.12772 9.999e-05
FALSE Residuals 36 27.0894 0.75248 0.87228
FALSE Total 38 31.0557 1.00000
FALSE
FALSE otu.df.tong.lungsamp$Sample_Type ***
FALSE Residuals
FALSE Total
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.tl ~ adonis.otudf.tl$Sample_Type, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.tl$Sample_Type 1 1.039 1.03903 1.5005 0.05265 0.0133 *
FALSE Residuals 27 18.696 0.69244 0.94735
FALSE Total 28 19.735 1.00000
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
FALSE
FALSE Call:
FALSE adonis(formula = adonis.hel.tb ~ adonis.otudf.tb$Sample_Type, permutations = 10000, method = "euclidean")
FALSE
FALSE Permutation: free
FALSE Number of permutations: 10000
FALSE
FALSE Terms added sequentially (first to last)
FALSE
FALSE Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
FALSE adonis.otudf.tb$Sample_Type 1 2.6012 2.60116 3.2809 0.10489 9.999e-05 ***
FALSE Residuals 28 22.1989 0.79282 0.89511
FALSE Total 29 24.8001 1.00000
FALSE ---
FALSE Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1V. Diversity Indices
5.1 Alpha Diversity
FALSE Tukey multiple comparisons of means
FALSE 95% family-wise confidence level
FALSE
FALSE Fit: aov(formula = tukey_otu_df[, "Unique_Otus_per_1k_reads"] ~ tukey_otu_df[, "Alpha_Div_Groups"])
FALSE
FALSE $`tukey_otu_df[, "Alpha_Div_Groups"]`
FALSE diff lwr upr
FALSE Isolation Control-BAL -10.612291 -27.006526 5.7819429
FALSE Lung-BAL 25.049970 7.457534 42.6424053
FALSE Sampling Control-BAL -2.528455 -20.690372 15.6334619
FALSE Sequencing Control-BAL -13.515401 -27.202081 0.1712778
FALSE Lung-Isolation Control 35.662261 18.778539 52.5459833
FALSE Sampling Control-Isolation Control 8.083837 -9.392476 25.5601491
FALSE Sequencing Control-Isolation Control -2.903110 -15.666004 9.8597843
FALSE Sampling Control-Lung -27.578425 -46.183380 -8.9734692
FALSE Sequencing Control-Lung -38.565371 -52.834721 -24.2960216
FALSE Sequencing Control-Sampling Control -10.986946 -25.952766 3.9788736
FALSE p adj
FALSE Isolation Control-BAL 0.3746804
FALSE Lung-BAL 0.0014826
FALSE Sampling Control-BAL 0.9950044
FALSE Sequencing Control-BAL 0.0545862
FALSE Lung-Isolation Control 0.0000011
FALSE Sampling Control-Isolation Control 0.6949822
FALSE Sequencing Control-Isolation Control 0.9684857
FALSE Sampling Control-Lung 0.0008519
FALSE Sequencing Control-Lung 0.0000000
FALSE Sequencing Control-Sampling Control 0.2510366
FALSE Tukey multiple comparisons of means
FALSE 95% family-wise confidence level
FALSE
FALSE Fit: aov(formula = tukey_otu_df[, "Shannon"] ~ tukey_otu_df[, "Alpha_Div_Groups"])
FALSE
FALSE $`tukey_otu_df[, "Alpha_Div_Groups"]`
FALSE diff lwr upr p adj
FALSE Isolation Control-BAL -0.9947478 -1.8514868 -0.1380087 0.0147639
FALSE Lung-BAL 0.6346623 -0.2846930 1.5540176 0.3098538
FALSE Sampling Control-BAL -0.5859416 -1.5350572 0.3631740 0.4234102
FALSE Sequencing Control-BAL -1.2226705 -1.9379166 -0.5074244 0.0000876
FALSE Lung-Isolation Control 1.6294101 0.7470911 2.5117290 0.0000205
FALSE Sampling Control-Isolation Control 0.4088062 -0.5044807 1.3220931 0.7201786
FALSE Sequencing Control-Isolation Control -0.2279227 -0.8948932 0.4390477 0.8732743
FALSE Sampling Control-Lung -1.2206039 -2.1928720 -0.2483357 0.0067338
FALSE Sequencing Control-Lung -1.8573328 -2.6030284 -1.1116372 0.0000000
FALSE Sequencing Control-Sampling Control -0.6367289 -1.4188211 0.1453633 0.16378335.2 Beta Diversity
FALSE
FALSE Pairwise comparisons using Wilcoxon rank sum test with continuity correction
FALSE
FALSE data: bray_dist_lbtce_long_mut_filt$BC_Index and bray_dist_lbtce_long_mut_filt$Sample_Type
FALSE
FALSE BAL Blank Cecum Lung
FALSE Blank 0.0074 - - -
FALSE Cecum < 2e-16 < 2e-16 - -
FALSE Lung < 2e-16 3.7e-11 < 2e-16 -
FALSE Tongue < 2e-16 < 2e-16 < 2e-16 0.0001
FALSE
FALSE P value adjustment method: BH
FALSE
FALSE Wilcoxon rank sum exact test
FALSE
FALSE data: BC_Index by Comparison_Type
FALSE W = 85, p-value = 0.0004114
FALSE alternative hypothesis: true location shift is not equal to 0VI. Relative Abundance
